Objective To establish the assay method of hydroxyproline(HYP) level in rat lung tissue for evaluating the lung fi-brosis degree .Methods The impact of different acid hydrolyzable time ,oxidative time ,developing time on the assay results of the HYP level in rat lung tissue was studied .On this basis ,the assay method for determining the HYP level was established and prelim-inarily applied in the detection of the HYP level in rat lung tissue .Results The optimal condition for the HYP level detection in rat lung tissue was as follows :7 .50 mol HCL was hydrolyzed for 16 h at 110 ℃ ,oxidized for 10 min at room temperature and devel-oped for 25min at 60℃ .The sensitivity of this method was 0 .067μg/mL .The recovery rate and the average CV of this method were 88 .85% -110 .88% and 4 .70% -6 .60% ,respectively .In the study of bleomycin induced rat lung fibrosis ,the HYP level of the model group was obviously higher than that of the control group .Conclusion This method has high sensitivity ,high recovery rate and good reproducibility ,and may be used as a reliable quantitative method to judge the lung fibrosis level in clinic .

Objective: To identify the optimal placement of bone tunnel during near-isometric anterior cruciate ligament reconstruction (ACLR), and its dimensional relationship with knee anatomic structure after summarizing published researches. Methods: PubMed, Embase, and CNKI were screened for Chinese or English articles on clinical studies, cadaveric studies of knee and reviews on bone tunnel placement in anterior cruciate ligament near-isometric reconstruction and isometry of native anatomic fibers. Related articles were extracted and systematically reviewed. Results: A total of 21 articles were finally included after screening. This systematic review found that most of the literatures were cadaveric studies, among which three dimensional imaging techniques, combined with cadaveric studies, were commonly used as a method of determining the length change between spots on the tibial plateau and lateral condyle of femur in recent years. There were 3 case series and 1 prospective cohort study. On the side of lateral condyle of femur, Blumensaat's line, lateral intercondylar ridge and insertion of intact anterior cruciate ligament were commonly used as reference to describe the near isometric area, while on the side of tibial plateau, the anterior horn of lateral meniscus were used as reference for reconstruction. The bone tunnel on tibial side was ignored compared with the femoral side when considering the isometry of reconstruction. There were overlaps among areas of bone tunnel placement found by different studies in near-isometric reconstruction. Not all native anterior cruciate ligament fibers were near-isometric. Conclusion The present study found that the area of femoral bone tunnel of anterior cruciate ligament near-isometric reconstruction were near the anterior part of lateral intercondylar ridge, deeply positioned with longitudinal axis close to the extension line of posterior cortical part of femur, coinciding with the distribution of direct fibers. There was contradiction on whether anterior cruciate ligament near-isometric reconstruction areas and anterior cruciate ligament anatomic insertion sites overlapped with each other. The most isometric area within the anatomic insertion site was the area of anterior fibers near lateral intercondylar ridge, which carried a large portion of the total anterior cruciate ligament load. Generally, there was a scarce of clinical researches in the field of isometry of ACLR. For the purpose of attesting the accuracy of the results we found, a combination of intra-operative observation on the displacement of graft relative to bone-tunnel and post-operative imaging such as MRI and three dimensional CT should be considered.

Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).

AIM:To evaluate the effects of treatment with HP 1188-immunoglobulin yolk ( HP1188-IgY) on Helicobacter pylori ( H.pylori)-infected gastritis in BALB/c mice.METHODS:BALB/c mice were used to establish an animal model of H.pylori-infected gastritis, and the mice were divided into 8 groups (10 mice per group).Oral antibiotics were used in group 1, 1 mg HP1188-IgY in group 2, 1 mg HP1188-IgY plus 30%sucralfate in group 3, 5 mg HP1188-IgY in group 4, 5 mg HP1188-IgY plus 30%sucralfate in group 5, PBS in group 6, and 30% sucralfate in group 7 with the treatment once per day for 10 d;and 2.5 mg HP1188-IgY was injected hypodermically twice with a 48-h interval in group 8.Another 10 mice were used as normal control in group 9.The planting of bacteria in the stomach was assayed by bacteri-al culture, rapid urease test, PCR and pathological sectioning .RESULTS:Intragastric administration with 1 mg HP1188-IgY plus 30%sucralfate per day effectively cured the injury of gastric mucosa caused by H.pylori infection, and the effect has no significant difference compared with antibiotics (P>0.05).CONCLUSION:We establish a BALB/c mouse mod-el infected with H.pylori successfully.Sucralfate (30%) is an ideal protectant for HP1188-IgY, which might decrease H. pylori infection in the stomach of BALB/c mice by oral inoculation .

Objective To synthesize and identify artificial antigens of lung elastin degradation peptide and for the purpose of preparation of COPD test.Methods The artificial antigens were synthesized by Sulfo-SMCC and KLH.The complete antigens were identified by ultraviolet spectrum and SDS-PAGE.Immunize Balb/c mice was used to prepare antibody.The antiserum activity was evaluated by indirect competitive ELISA.Results The artificial antigens were identified by ultraviolet spectrum and SDS-PAGE. The protein concentration was 1.181 mg/mL.The titer of antiserum was 1∶64 000,and IC50 was 13.7 ng/mL.The antiserum had no cross-reaction with nonsense peptide.Conclusion The artificial antigens were acquired successfully,which had good immunoge-nicity.The results have laid basis for COPD test.

Objective To investigate the effects of lincRNA-cox2 on the polarization of murine RAW264. 7 macrophages by analyzing the expression of lincRNA-cox2 in RAW264. 7 macrophages of M1 and M2 phenotypes. Methods Murine RAW264. 7 cells were induced by IFN-γand LPS to polarize to M1 phenotype, and were induced by IL-4 to polarize to M2 phenotype. The expression of lincRNA-cox2 in M1 and M2 macrophages were analyzed by real-time quantitative PCR ( RT-PCR) . We designed and synthesized siRNA oligo for lincRNA-cox2 and unrelated sequences. Then the siRNA oligo and NC oligo were transfected into RAW264. 7 cells by LipofectmineTM 2000. The transfected RAW264. 7 cells were induced by IFN-γand LPS or by IL-4 to polarize to M1 or M2 macrophages. Enzyme linked immunosorbent assay ( ELISA) was performed to measure the secretion of IL-10 and IL-12 induced in different conditions. The expression of in-ducible nitric oxide synthase ( iNOS ) , TNF-α, arginase 1 ( Arg-1 ) and found in inflammatory zone 1 (Fizz1) at mRNA level were detected by RT-PCR. The M1 macrophages were transfected with siRNAs to knock down the expression of lincRNA-cox2 for analyzing the biological effects of lincRNA-cox2 on the polar-ization of macrophages. Results The relative expression of lincRNA-cox2 in M1 macrophages was signifi-cantly higher than that in RAW264. 7 cells and M2 macrophages. Compared with the control group, the RAW264. 7 cells transfected with lincRNA-cox2-siRNA showed decreased secretion of IL-12 and inhibited expression of iNOS and TNF-αat mRNA level after IFN-γand LPS induction, but increased secretion of IL-10 and enhanced expression of Arg1 and Fizz1 at mRNA level after IL-4 induction. Transfecting the M1 mac-rophages with lincRNA-cox2-siRNA inhibited the secretion of IL-12, but promoted the secretion of IL-10. Conclusion This study indicated that lincRNA-cox2 was involved in the regulation of macrophage pheno-types by promoting the polarization to M1 macrophages and inhibiting the polarization to M2 macrophages.

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Objective: To assess plantar arch index (AI) and prevalence of flat feet in school-age children (7-12 years old) in Shanghai and evaluate the relationship between flat feet with age, gender, weight status (BMI) and occurring sides, and to provide a reference for the prevention of flatfeet. Methods: Three-dimensional foot measuring instruments were used to measure bilateral foot length, medial arch height, AI and arch height ratio (AHR) in school-age children in Shanghai. Statistical analysis of these foot parameters was performed. Results: A total of 3 226 children between aged 7 and 12 in Shanghai were measured with AI of (0.27±0.05) and AHR of (3.02±1.89). Prevalence of flat feet in the group of 7 to 12 year old children was 56.1%. Prevalence of flatfeet decreased significantly with age: 72.6% at 7 years old and 37.9% at 12 years old. Boys had a significantly greater risk for flat feet than girls: the prevalence of flat feet was 62.9% for boys and 47.8% for girls (OR=1.81, 95%CI=1.57-2.10). This risk was independent of age but related to gender. The risk of flat feet in boys was always higher than that in girls at every age. For children aged 7-8, being overweight was not significantly related to the occurrence of flat feet. However, for children aged 9-12 who were overweight were more likely to have flat feet than those of normal weight. The OR increased with age: from 1.44 (95%CI=1.03-2.03) at 9 to 2.96 (95%CI=1.68-5.23) at 12. There was no difference on which side flat feet would occur (χ2=0.95，P=0.33). Conclusion This finding shows that prevalence of flat feet is influenced by age, gender and weight status. AI and prevalence of flat feet in children aged 7-12 decreases with age, and boys have significantly higher prevalence of flat feet than girls. Overweight children aged 9 or older have a higher risk of flat feet.

Objective To evaluate the clinical effect of milrinone nebulized inhalation for improving intraoperative cardiac function and pulmonary arterial pressure in the patients with chronic obstructive pulmonary disease (COPD) complicating pulmonary hypertension (PH).Methods Forty-four surgical patients with COPD complicating PH in the Chongqing Municipal Medical Emergency Center from June 2015 to June 2016 were chosen,including 23 cases of thoracic surgery,13 cases of abdominal surgery and 8 cases of lower extremity fracture surgery.The patients were divided into the control group and treatment group,22 cases in each group.The control group received the routine comprehensive treatment.In addition receiving the conventional comprehensive treatment,milrinone nebulized inhalation in the treatment group was given before general anesthesia.Both of the two groups were imbedded with floating catheter in the right internal jugular vein for detecting the clinical indexes:cardiac output (CO),pulmonary artery systolic pressure (PASP),pulmonary artery mean pressure (PAMP) and pulmonary capillary wedge pressure (PCWP).Results Compared with before treatment,CO after treatment in the treatment group was significantly increased (P＜0.05),PASP,PAMP and PCWP after treatment in the treatment group were significantly decreased(P＜0.05).CO,PASP,PAMP and PCWP in the control group had no statistical difference between before and after treatment,the difference was not statistically significant (P＞0.05).Conclusion Intraoperative milrinone nebulized inhalation in the patients with COPD complicating PH can effectively improve the patient's cardiopulmonary function.

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.
Apoptosis ; radiation effects ; Electromagnetic Fields ; Hep G2 Cells ; Humans ; Membrane Potential, Mitochondrial ; physiology ; radiation effects ; Time Factors